Bacterial nodulation protein NodZ is a chitin oligosaccharide fucosyltransferase which can also recognize related substrates of animal origin

Abstract

The nodZ gene, which is present in various soil bacteria such as Bradyrhizobium japonicum, Azorhizobium caulinodans, and Rhizobium loti, is involved in the addition of a fucosyl residue to the reducing N-acetylglucosamine residue of lipochitin oligosaccharide (LCO) signal molecules. Using an Escherichia coli strain that produces large quantities of the NodZ protein of B. japonicum, we have purified the NodZ protein to homogeneity. The purified NodZ protein appears to be active in an in vitro transfucosylation assay in which GDP-␤-fucose and LCOs or chitin oligosaccharides are used as substrates. The products of the in vitro reaction using chitin oligosaccharides as substrate were studied by using mass spectrometry, linkage analysis, and composition analysis. The data show that one fucose residue is added to C6 of the reducing-terminal N-acetylglucosamine residue. The substrate specificity of NodZ protein was analyzed in further detail, using radiolabeled GDP-␤-fucose as the donor. The results show that chitin oligosaccharides are much better substrates than LCOs, suggesting that in Rhizobium NodZ fucosylates chitin oligosaccharides prior to their acylation. The free glycan core pentasaccharides of N-linked glycoproteins are also substrates for NodZ. Therefore, the NodZ enzyme seems to have an activity equivalent to that of the enzyme involved in the addition of the C6-linked fucosyl substituent in the glycan core of N-linked glycoproteins in eukaryotes. Oligosaccharides that contain only one N-acetylglucosamine at the reducing terminus are also substrates for NodZ, although in this case very high concentrations of such oligosaccharides are needed. An example is the leukocyte antigen Lewis-X, which can be converted by NodZ to a novel fucosylated derivative that could be used for binding studies with E-selectin.The symbiotic relationship between legumes and rhizobia (i.e., Rhizobium, Bradyrhizobium, or Azorhizobium) can result in the formation of a nitrogen-fixing root organ, the nodule. The development of legume nodules is largely controlled by reciprocal signal exchange between the symbiotic partners. Legume roots secrete specific flavonoids or isoflavonoids that induce the transcription of many bacterial genes governing the early steps of this interaction (nod, nol, and noe genes). Many of these genes are involved in the synthesis and secretion of signal molecules, which are lipochitin oligosaccharides (LCOs). The chitin oligosaccharide backbone of all LCOs (also known as Nod factors) is N-acylated on the non-reducing-terminal residue. The basic structure of LCOs is synthesized by the cooperative action of NodA, NodB, and NodC (1-6). Additional gene products provide chemical decorations which, in some cases, have been shown to determine host specificity (for reviews see refs. 7-11). For instance, in a recent study (12) it was shown that transfer of the Bradyrhizobium japonicum nodZ gene to Rhizobium leguminosarum biovar viciae leads to the biosynthesis of LCOs that are fucosylated on C6 of the reducing-...

Molecular Basis of Symbiotic Promiscuity

“…Perhaps this suggests that both arabinosylated and fucosylated Nod factors are (nodZ, nolK). Fucose is a frequent substitution, and its addition to Nod factors is encoded by nodZ in many rhizobia (162,171,208,210,261). Since all the usual Glycine max symbionts (B. elkanii, B. japonicum, R. fredii, and strain NGR234) secrete fucosylated Nod factors, the expectation that mutation of nodZ would prevent the nodulation of soybeans was strong.…”

Identification of Essential Amino Acids in theAzorhizobium caulinodansFucosyltransferase NodZ

“…The metal cation requirement of recombinant NodZ was assessed by performing reactions in the presence of 10 mM concentrations of various divalent cations or EDTA. Product of the enzyme reaction was identified in preliminary experiments by thin-layer chromatography as described elsewhere (46). Sequence analysis.…”

“…Fucose addition to Nod factors is encoded by nodZ in many rhizobia (31,45,46,52). Indeed, recombinant NodZ catalyzes the ␣1,6-fucosylation of the chitin oligosaccharide backbone (45,46).…”

“…Indeed, recombinant NodZ catalyzes the ␣1,6-fucosylation of the chitin oligosaccharide backbone (45,46). Interestingly, this enzyme seems to have an activity comparable to eukaryotic enzymes that fucosylate the chitobiosyl core of complex-type N-linked oligosaccharide (46). Fucosyltransferases (FucTs) are enzymes catalyzing the transfer of fucose from GDP-fucose to various oligosaccharide acceptor substrates.…”

“…Many fucosyltransferase genes have been cloned in humans and in various animal species (13,36), as have a few genes from invertebrates (15,29,56) and plants (19,28,42). In prokaryotes, in addition to the cloned bacterial nodZ genes from Rhizobium species (45,46), several studies have reported the cloning and expression of fucosyltransferase genes from the pathogenic bacterium Helicobacter pylori (23,30,61). All fucosyltransferases that have been cloned to date transfer fucose either in a ␣1,2-linkage to a galactose residue, or in ␣1,3-, ␣1,4-, or ␣1,6-linkage to a GlcNAc residue.…”